HIV-1 protease, which is required for virion maturation and proliferation, exists naturally as a homodimeric aspartyl protease in which each monomer provides an active site aspartic acid. As expected, site- directed mutation in which the catalytic Asp25 is changed to Asn results in a completely inactive protein complex. In tissue culture experiments, Asp25Asn acted as a dominant negative inhibitor and prevented proteolytic processing and maturation of the HIV virion. A second generation of defective monomers was created to improve the strength of the dominant negative inhibitor. Several structures of protease complexed with inhibitor were overlaid to map the substrate binding pocket. Computational modeling predicted that replacement of Asp25 with lysine or arginine and Gly49 and Ile50 with tryptophans in the variant monomer would fill subsites of the substrate binding pocket on the wild type monomer. Bulky new residues would not only stabilize the heterodimer but also discourage formation of variant homodimers. In vivo analysis of these variants has demonstrated their improved ability to inhibit both polyprotein processing in and maturation of HIV-1 virions. My project is currently focused on further strengthening the heterodimer interface by screening randomizedlibraries of variants using a phage display system as well as by rational design of new variants. Computer-assisted design is based on comparisons of recent, high-resolution structures of HIV protease as well as on MD relaxation of a heterodimer between the Asp25Lys/Gly49Trp/Ile50Trp variant and wild type. Variants resulting from either combinatorial selection or rational that are shown to form stronger heterodimers with wild type protease will be modeled using MidasPlus in the Computer Graphics Laboratory. The necessary systems for analysis of the consequences of HIV protease mutations both on the stability of the dimer in vitro and on physiologically relevant activity in vivo have been established in the Craik lab. The development of HIV-1 protease variants that aggressively form heterodimers with wild type monomers presents a potential new class of macromolecular drugs that may be highly effective against a variety of strains of HIV, including protease-resistant strains.